First report of Ilyonectria robusta causing root rot on Coptis chinensis in Chongqing, China.

Abstract

Coptis chinensis belongs to the Ranunculaceae family and is a widely used traditional Chinese herb. Chongqing Municipality produces >60% of China's production. Root rot seriously reduced yield and quality (Mei et al. 2021). In May 2020, root rot of C. chinensis were observed on 3-year-old roots with an average incidence of 45.3% in three commercial fields (about 0.5 acre) in Fengmu Town, Shizhu County (30.24°N; 108.48°E) from Chongqing. Diseased plants were stunted and less vigorous with wilting and twisting leaves. Brown or black discoloration lesion was appeared in the vascular and cortical tissue of roots and rhizomes. Ten fresh symptomatic plants were randomly sampled from the fields. Root tissues were surface sterilized in 75% ethanol for 60s, rinsed thrice with sterile water, placed on potato dextrose agar (PDA), and incubated at 25°C for 7 days. A total of 11 isolates were obtained from the infected tissues. Pure colonies of all fungal isolates had similar characteristics, and five isolates (a2, a4, a9, a11, a12) were randomly selected for further study. Colonies of this fungus were aurantium and felty at first, and then became brownish grey. Macroconidia (n=50) were predominating, hyaline, cylindrical, predominantly straight with both ends broadly rounded, 1~3 septate; one septate, 18.8~25.5×5.9~6.8μm; two septate, 22.6~35.4×6.1~7.2μm; three septate, 26.1~42.5×7.2~8.0 μm. Microconidia (n=50) were hyaline, ellipsoid to ovoid, 0 to 1 septate; aseptate, 7.5~8.8×3.4~4.3μm. Chlamydospores (n=50) were hyaline at first, and becoming brown, globose to subglobose, smooth, 8.3~12.5×8.1~13.5μm, mostly occurring intercalary in chains. The DNA of isolates were extracted and the ITS, HIS, TEF, TUB2 genes were amplified and sequenced using the primers ITS1/ITS4, CYLH3F/CYLH3R, EF1/EF2, T1/CYLTUB1R, respectively (Cabral et al. 2012). The representative isolate a2 were deposited in GenBenk (OK105140, ITS; OM799544, HIS; OK493444, TEF; OK493445, TUB2). BLAST analysis showed the ITS, HIS, TEF, TUB2 sequences of a2 were 100% (417/417), 100% (472/472), 100% (762/762), and 99.7% (490/491) homology with those of Ilyonectria robusta (CBS 605.92) from Tilia petiolaris in Germany. Phylogenetic analysis using Maximum Likelihood and concatenated sequences (ITS+HIS+TEF+TUB2) with MEGA7 placed isolate a2 in I. robusta with 100% bootstrap support. The isolate was thus identified as I. robusta based on morphological and molecular characteristics (Cabral et al. 2012). Thirty healthy 6-month-old C. chinensis plants were used for the pathogenicity tests, and five plants were into each of 6 pots. 10ml of conidia suspension (1×106conidia/ml) of 10-day-old isolate a2 was gently applied to the soil in each of 6 pots. Sterile water (10ml) was applied to each of 6 pots as control. All 12 pots were placed in a greenhouse (25°C, 12h photoperiod). After 6 weeks inoculation, all inoculated plants showed twisting and wilting symptoms, and the roots showed light-brown to dark-brown lesions. No symptoms were observed on the controls. The pathogen was reisolated from all symptomatic roots and identified as I.robustaas previously described above. The test was repeated twice with similar results. Although this fungus was previously reported to cause root disease on many plants (Zheng et al. 2022; Qiao et al. 2019; Guggenheim et al. 2019), this is the first report of I. robusta causing root rot on C. chinensis in China, and will establish a foundation for controlling the disease.